JP2017507123A - Use of odontogenic stem cells and genetically modified odontogenic stem cells - Google Patents

Abstract

The present invention may be used to treat periodontal disease, repair periodontal bone or soft tissue defects, promote periodontal tissue regeneration, and / or acute and chronic bone tissue damage (eg, fractures). ) Or the use of odontogenic stem cells and genetically modified odontogenic stem cells in the preparation of a product for treating bone tissue defects. The present invention also provides an odontogenic stem cell into which an exogenous hepatocyte growth factor gene has been introduced by an adenovirus or an adeno-associated virus vector and / or genetically to obtain a genetically modified odontogenic stem cell. Compositions comprising the modified odontogenic stem cells are also provided.

Description

  The present invention relates to the use of odontogenic stem cells (dental pulp stem cells, stem cells collected from human deciduous deciduous dental pulp, periodontal ligament stem cells, apical stem cells) and genetically modified odontogenic stem cells, in particular periodontal Preparation of products to treat disease, repair periodontal bone or soft tissue defects due to various reasons (eg periodontal disease), and / or promote regeneration of periodontal tissue, and It relates to the use of odontogenic stem cells and genetically modified odontogenic stem cells in the preparation of a product for treating acute and chronic bone tissue damage (eg fractures) or bone tissue defects. The invention also relates to genetically modified odontogenic stem cells and compositions comprising odontogenic stem cells and / or genetically modified odontogenic stem cells.

  Periodontal disease is a common clinical oral disease, an infection with characteristic periodontal tissue damage (including periodontal ligament, cementum, bone, and gums), the main clinical symptoms of which are Gingival inflammation and bleeding, periodontal pocket formation, alveolar bone resorption, tooth relaxation, and even tooth wobble. Periodontal disease is not only a major cause of tooth loss, but is also associated with the occurrence of certain systemic diseases. Periodontal disease is prevalent in many people. In infants and adolescents, the prevalence of gingivitis is 70% to 90%, chronic periodontal disease is over 60%, invasive periodontal disease is 5% to Periodontal disease accounts for 30-44% of tooth loss. The best method after periodontal attachment disruption and alveolar bone development is to completely rebuild healthy periodontal tissue. Currently, clinical methods for treating periodontal disease include basic treatment of periodontal (tooth scaling, curettage, root formation), periodontal flap surgery, and periodontal tissue regeneration. The latest research methods at home and abroad to regenerate periodontal tissue include: (1) guided tissue regeneration GTR: growth of epithelial tissue into periodontal defects To avoid this, an absorbable or non-absorbable biofilm or titanium film is placed in the defect of the periodontal tissue. A filler, artificial bone, carrier, or periodontal regeneration inducing factor can be implanted into the periodontal defect. (2) In the engineering regeneration technology of periodontal tissue, research on obtaining seed cells is active. The main stem cells currently found to be associated with teeth include the following: dental pulp stem cells (DPSC), stem cells from human exfoliated deciduous teeth, SHED: SHED), periodontal ligament stem cells (PDLSC), apical stem cells (stem cells from apical papilla: SCAP). The overall study of these cells can not only have a positive effect on tooth development and growth, but also discover a source of seed tissue engineering seed cells.

  The inventors have surprisingly found that through numerous experiments, various sources of odontogenic stem cells have been used to repair periodontal bone and soft tissue defects due to various reasons (eg, periodontal disease). And that it can be used to promote periodontal tissue regeneration. At the same time, the inventors have discovered that various sources of dental stem cells can also be used to repair bones other than alveolar bone, and genetically modified odontogenic stem cells, particularly hepatocyte growth It was proved that the therapeutic effect by stem cells in which the factor (HGF) gene was modified was excellent.

  The first aspect of the present invention is to prevent and / or treat periodontal disease, repair periodontal bone or soft tissue defects caused by periodontal disease, and / or promote periodontal tissue regeneration. The use of odontogenic stem cells in the preparation of a product for

  A second aspect of the present invention is a dental preparation in the preparation of a product for preventing and / or treating acute and chronic bone tissue damage (eg, fractures) or bone tissue defects, or for promoting bone tissue repair. Relates to the use of sex stem cells.

  For use in the first and second aspects of the present invention, the odontogenic stem cells are selected from the group consisting of dental pulp stem cells, stem cells collected from human deciduous deciduous dental pulp, periodontal ligament stem cells, and apical stem cells At least one, for example, two.

  In one embodiment of the invention, the odontogenic stem cell is a dental pulp stem cell.

  In another embodiment of the invention, the odontogenic stem cells comprise dental pulp stem cells and periodontal ligament stem cells.

  A third aspect of the invention relates to genetically modified odontogenic stem cells that express exogenous hepatocyte growth factor.

  The genetically modified odontogenic stem cell according to any of the third aspects of the present invention is a group consisting of dental pulp stem cells, stem cells collected from human fallen deciduous dental pulp, periodontal ligament stem cells, and apical stem cells At least one selected from, for example, two.

  In one embodiment of the invention, the odontogenic stem cell is a dental pulp stem cell.

  In another embodiment of the invention, the odontogenic stem cells comprise dental pulp stem cells and periodontal ligament stem cells.

  The genetically modified odontogenic stem cell in any of the third aspects of the present invention is introduced with an exogenous hepatocyte growth factor gene and expresses the exogenous hepatocyte growth factor.

  In the genetically modified odontogenic stem cell according to any of the third aspects of the present invention, an exogenous hepatocyte growth factor gene is introduced by an adenovirus or an adeno-associated virus vector.

  The fourth aspect of the present invention is to prevent and / or treat periodontal disease, repair periodontal bone or soft tissue defects caused by periodontal disease, and / or promote periodontal tissue regeneration. The use of odontogenic stem cells according to any of the third aspects of the invention in the preparation of a product for

  A fifth aspect of the present invention relates to the preparation of a product for preventing and / or treating acute and chronic bone tissue damage (eg fractures) or bone tissue defects, or for promoting the repair of bone tissue. The use of odontogenic stem cells according to any of the third aspects of the invention.

  The present invention also relates to a composition comprising an effective amount of odontogenic stem cells, and optionally a pharmaceutically acceptable carrier or excipient. The composition is acute for preventing and / or treating periodontal disease, for repairing periodontal bone or soft tissue defects caused by periodontal disease, and / or for promoting periodontal tissue regeneration. And used to prevent and / or treat chronic bone tissue damage (eg, fractures) or bone tissue defects, or to promote bone tissue repair.

  In one embodiment of the invention, the composition is a cell suspension comprising odontogenic stem cells.

  Preferably, the odontogenic stem cells are at least one selected from the group consisting of dental pulp stem cells, stem cells collected from human fallen deciduous dental pulp, periodontal ligament stem cells, and apical stem cells, for example, two types.

  In one embodiment of the invention, the odontogenic stem cell is a dental pulp stem cell.

  In another embodiment of the invention, the odontogenic stem cells comprise dental pulp stem cells and periodontal ligament stem cells.

  The invention also relates to a composition comprising an effective amount of odontogenic stem cells of the third aspect of the invention and optionally a pharmaceutically acceptable carrier or excipient.

  In one embodiment of the invention, the composition is a cell suspension comprising odontogenic stem cells of the third aspect of the invention.

  The present invention relates to a product for preventing and / or treating periodontal disease, repairing a periodontal bone or soft tissue defect caused by periodontal disease, and / or promoting regeneration of periodontal tissue. It also relates to the use of the composition of the invention in preparation.

  The invention relates to the use of the composition of the invention in the preparation of a product for preventing and / or treating acute and chronic bone tissue damage (eg fractures) or bone tissue defects or for promoting the repair of bone tissue. Also related.

  The present invention is directed to prevent and / or treat periodontal disease, repair periodontal bone or soft tissue defects due to periodontal disease, and / or promote periodontal tissue regeneration, It also relates to the use of hepatocyte growth factor in the preparation of a product for preventing and / or treating chronic bone tissue damage (eg, fractures) or bone tissue defects, or to promote bone tissue repair.

  The present invention relates to a method for preventing and / or treating periodontal disease, repairing a periodontal bone or soft tissue defect, and / or promoting regeneration of periodontal tissue. A method comprising administering an effective amount of odontogenic stem cells or genetically modified odontogenic stem cells in any of the third aspects, or any composition of the present invention to a subject in need thereof. Also related.

  The present invention is a method for preventing and / or treating acute and chronic bone tissue damage or bone tissue defect, or for promoting bone tissue repair, according to any of the third aspects of the present invention. It also relates to a method comprising administering an effective amount of odontogenic stem cells or genetically modified odontogenic stem cells, or any composition of the invention to a subject in need thereof.

  In one embodiment of the present invention, the odontogenic stem cells are at least one selected from the group consisting of dental pulp stem cells, stem cells collected from human deciduous deciduous dental pulp, periodontal ligament stem cells, and apical stem cells, for example, There are two types.

  In one embodiment of the present invention, cell suspension injection is employed for the treatment described above.

  The invention also provides acute and chronic bone tissue damage or bone to prevent and / or treat periodontal disease, to repair periodontal bone or soft tissue defects, to promote periodontal tissue regeneration. It also relates to odontogenic stem cells used to prevent and / or treat tissue defects and / or to promote bone tissue repair.

  In one embodiment of the present invention, the odontogenic stem cells are at least one selected from the group consisting of dental pulp stem cells, stem cells collected from human deciduous deciduous dental pulp, periodontal ligament stem cells, and apical stem cells, for example, There are two types.

  The present invention relates to acute and chronic bone tissue defects or bone tissue defects to prevent and / or treat periodontal disease, to repair periodontal bone or soft tissue defects, to promote periodontal tissue regeneration. It also relates to odontogenic stem cells in the third aspect of the invention used to prevent and / or treat and / or promote bone tissue repair.

  In an embodiment of the present invention, the odontogenic stem cells are at least one selected from the group consisting of dental pulp stem cells, stem cells collected from human deciduous deciduous dental pulp, periodontal ligament stem cells, and apical stem cells, for example, 2 It is a seed.

  The present invention is described in detail as follows. First, in the present invention, the effects of different odontogenic stem cell sources on the treatment of periodontal bone and soft tissue and the regeneration of periodontal tissue were examined. Accordingly, the first aspect of the present invention is for treating periodontal disease, repairing periodontal bone or soft tissue defects resulting from periodontal disease, and / or promoting periodontal tissue regeneration. The use of odontogenic stem cells in the preparation of products.

  In the present invention, first, odontogenic stem cells obtained from different tissues (for example, pulp, periodontal membrane, apex) are isolated and cultured to detect surface markers and osteogenic differentiation of the obtained odontogenic stem cells. These cells were confirmed to be mesenchymal stem cells by adipogenic differentiation. Therefore, the obtained odontogenic stem cells are used as cell suspensions, and odontogenic stem cells from different sources treat favorable periodontal bone and soft tissue defects and regenerate periodontal tissues. Experimentally proved to have

  At the same time, the inventor has verified that odontogenic stem cells obtained from different sources can be used to repair other bone tissue damage in addition to periodontal bone. Accordingly, a second aspect of the present invention provides odontogenic stem cells in the preparation of products for treating acute and chronic bone tissue damage (eg, fractures) or bone tissue defects, or for promoting bone tissue repair. About the use of.

  The present invention is a composition comprising an effective amount of odontogenic stem cells for treating periodontal disease, repairing periodontal bone or soft tissue defects caused by periodontal disease, It relates to said composition used for promoting regeneration, for treating acute and chronic bone tissue damage (eg fractures) or bone tissue defects and / or for promoting bone tissue repair.

  At the same time, the present inventor treats periodontal disease because odontogenic stem cells lose their special culture environment after living transplantation and get nutrition only through permeation microcirculation in the range of 100-200 μm. For this reason, it has also been found that there are some limitations to the local administration of odontogenic stem cells. Over 90% of the cells die soon after transplantation due to undernourishment, after which the cells cannot successfully exert their anti-inflammatory and anti-apoptotic effects and promote proliferation and other functions.

  Hepatocyte growth factor (HGF) is a multifunctional growth factor that plays a leading role in promoting angiogenesis, inhibiting fibrosis, reducing cell apoptosis and anti-inflammatory effects in vivo, and the like. The present inventor has discovered that HGF has the following physiological functions in the process of periodontal tissue repair: (1) soluble intercellular adhesion molecule -1 (sICAM-1) (2) Inhibiting cell apoptosis, in particular, activating its PI3K / Akt signaling pathway or SPK-S1P signaling pathway to exert its anti-apoptotic effect; (3) Vascular Improve blood reflux by improving endothelial cells and angiogenesis, improving local blood supply and hypoxia generation. However, the structure of the HGF protein is complex and is rapidly metabolized in vivo. In order to obtain high concentrations of HGF in the area of local injury, it is necessary to carry out continuous large doses of the recombinant protein. Thus, using gene therapy strategies to treat periodontal disease or other systemic bone tissue damage, and using odontogenic stem cells modified with recombinant adenovirus with HGF It has the dual advantage of therapy and cell growth factor therapy and has a synergistic effect. On the other hand, by local injection, most odontogenic stem cells are retained or homed in the damaged periodontal tissue or other bone tissue, and exert a stem cell repairing action, and at the same time, a high local concentration of HGF Exerts a biological role, that is, the high expression of HGF promotes the survival and proliferation of stem cells derived from teeth, thereby enhancing the therapeutic effect of odontogenic stem cells.

  Accordingly, a third aspect of the present invention relates to a genetically modified odontogenic stem cell characterized by expressing an exogenous hepatocyte growth factor. In one embodiment of the invention, the expression of HGF is secretory expression because the cell secretes hepatocyte growth factor out of the cell. In an embodiment of the present invention, by introducing an exogenous hepatocyte growth factor gene into an odontogenic stem cell, the odontogenic stem cell expresses the exogenous hepatocyte growth factor, and the odontogenic stem cell becomes an exogenous hepatocyte growth. Factor is expressed.

  The method of introducing an exogenous hepatocyte growth factor gene into odontogenic stem cells is a common method for introducing an exogenous gene into a cell, such as viral transfection, plasmid transfection, and liposome transfection. It is done. In one embodiment of the invention, the method for introducing foreign genes into odontogenic stem cells is viral transfection, such as transfection of adenovirus or adeno-associated virus. In one embodiment of the invention, the virus is an adenovirus.

  In one embodiment of the present invention, HGF is a human hepatocyte growth factor, and its gene sequence is determined by K Miyazawa et al., Molecular cloning and sequence analysis of cDNA for human hepatocyte growth fator. Biochem Biophys Res Commun, 1989, 163 ( 2): 967-973.

  The preparation method of HGF-modified odontogenic stem cells of the present invention is as follows. Odontogenic stem cells (for example, dental pulp stem cells, stem cells collected from human deciduous deciduous dental pulp, periodontal ligament stem cells, and apical stem cells) were isolated and cultured. After culturing in vitro to the third generation, odontogenic stem cells were modified with hepatocyte growth factor (eg, recombinant adenovirus (AD-HGF) with human hepatocyte growth factor gene). 24-48 hours after modification, cell suspensions were collected for treatment by injection.

  Experimental results have shown that HGF-modified odontogenic stem cells can home to local sites of damaged periodontal tissue or other bone tissue and express HGF. HGF can exert anti-inflammatory effects, promote angiogenesis and other biological effects, as well as increase the survival rate and proliferation of transplanted odontogenic stem cells. Odontogenic stem cells can play a role in immune regulation and reduce inflammation in damaged tissues.

  Under the combined action of HGF and MSC, periodontal tissue damage can be effectively reduced, and regeneration of periodontal tissue and other bone tissues can be promoted, thereby achieving the therapeutic purpose.

  Therefore, the present invention treats periodontal disease, repairs periodontal bone or soft tissue defects caused by periodontal disease, promotes periodontal tissue regeneration, and acute and chronic bone tissue damage. Genetically modified odontogenic stem cells according to any of the third aspects of the invention in the preparation of a product for treating (eg fractures) or bone tissue defects and / or repairing bone tissue Also related to the use of.

  The invention also relates to a composition comprising an effective amount of odontogenic stem cells or genetically modified odontogenic stem cells according to any of the third aspects of the invention.

  In one embodiment of the present invention, the genetically modified odontogenic stem cell refers to an odontogenic stem cell in which HGF has been modified, that is, an odontogenic stem cell that expresses a large amount of HGF upon introduction of the HGF gene. .

  In one embodiment of the invention, the composition treats periodontal disease, periodontal bone tissue and soft tissue defects resulting from periodontal disease, promotes periodontal tissue regeneration, and acute and chronic bone. Used to treat tissue damage (eg, fractures) or bone tissue defects and / or to promote bone tissue repair.

  In one embodiment of the present invention, a method for treating periodontal bone defects using odontogenic stem cells or HGF-modified odontogenic stem cells is an experimental model of periodontal disease in the first mandibular molar of minipigs. Once established, a suspension of odontogenic stem cells or HGF genetically modified odontogenic stem cells is injected into the bone defect area. Laboratory tests, imaging, histology, and other indicators (including gingival crevice bleeding index, periodontal pocket depth, and loss of clinical periodontal attachment) are used to assess treatment effects.

  In one embodiment of the invention, odontogenic stem cells or HGF-modified odontogenic stem cells are used to treat acute and chronic bone tissue damage or defects, or to promote bone tissue repair. In this method, after establishing a mouse femoral shaft shaft fracture model, a suspension of odontogenic stem cells or HGF-modified odontogenic stem cells is injected into the fracture site, and the therapeutic effect is evaluated by image pattern analysis.

  Thus, the present invention treats periodontal disease, repairs periodontal bone or soft tissue defects, regenerates periodontal tissue, treats acute and chronic bone tissue damage or bone tissue defects And / or a method for promoting the repair of bone tissue comprising a therapeutically effective amount of odontogenic stem cells or genetically modified odontogenic stem cells according to any of the third aspects of the invention And a method comprising administering to a subject in need thereof. In an embodiment of the present invention, the odontogenic stem cells are at least one selected from the group consisting of dental pulp stem cells, deciduous deciduous dental pulp stem cells, periodontal ligament stem cells, and apical stem cells, for example, two types. In one embodiment of the present invention, cell suspension injection is used in the approaches described herein.

  In the present invention, the described odontogenic stem cell belongs to a mesenchymal stem cell that refers to a stem cell derived from an early developing mesoderm and ectodermal stem cell. They have inter alia the ability in pluripotency, hematopoietic support, promotion of stem cell transplantation, immune regulation, and self-renewal. They do not express hematopoietic stem cell surface markers such as CD14, CD31, CD34, CD45 and leukocyte differentiation antigen HLA-DR, but express surface markers such as CD44, CD29, CD90, CD105, CD73, CD166.

  In the present invention, odontogenic stem cells are derived from mammals. In one embodiment of the present invention, the odontogenic stem cell derived from a mammal is a group consisting of a human, a pig (for example, Mt. More selected.

  In the present invention, genetically modified odontogenic stem cells refer to odontogenic stem cells into which an exogenous hepatocyte growth factor gene has been introduced. Odontogenic stem cells express an exogenous hepatocyte growth factor gene.

  In the present invention, treating periodontal disease, repairing periodontal bone or soft tissue defects, promoting periodontal tissue regeneration, treating acute and chronic bone tissue damage or bone tissue defects, and / or Promoting bone tissue repair refers to treating autologous or allogeneic disease or regenerating tissue.

  In the present invention, periodontal disease includes gingivitis and periodontitis. The former occurs only in gingival tissue, while the latter is a chronic infection involving the four types of periodontal support tissues (gum, periodontal membrane, alveolar bone, and cementum), often periodontal Causes inflammation due to inflammation of the supporting tissues, the main clinical symptoms are gum inflammation, bleeding, periodontal pocket formation, alveolar bone absorption, alveolar bone height reduction, tooth wobble, tooth movement, There is a decrease in masticatory force, and there is a loss of teeth by applying force, or a natural loss of teeth.

  In the present invention, the periodontium refers to periodontal support tissue including gums, periodontal membrane, alveolar bone, and cementum.

  In the present invention, periodontal bone tissue includes alveolar bone and cementum.

  In the present invention, the periodontal soft tissue includes gingiva and periodontal membrane.

  In the present invention, periodontal bone tissue and soft tissue defects include bone and soft tissue defects due to chronic periodontitis, invasive periodontitis, necrotizing and ulcerative periodontitis.

  In the present invention, bone tissue is also understood as bone, which is a system that supports the human body, and mainly consists of bone, periosteum and bone marrow, and depending on the shape, long bone, short bone, flat bone, irregular bone, And can be classified as mixed bone.

  In the present invention, a bone tissue defect refers to a state in which the integrity of the bone structure has been destroyed. Trauma, infection, tumors, surgical debridement of osteomyelitis, and various congenital diseases are the main causes of bone defects.

  In one embodiment of the invention, the bone tissue injury is a fracture. In the present invention, unmodified means that HGF has not been modified, that is, exogenous HGF has not been introduced into the cell.

  In the present invention, the term “product” refers to various forms suitable for odontogenic stem cells to be used, for example, drugs, compositions, pharmaceutical compositions and the like.

  In the present invention, the term “composition” has a meaning generally understood by those skilled in the art, and each form usually used directly or indirectly (eg, predilution) in clinical practice, For example, it refers to a dosage form, a drug dosage form, a dosage form and the like. In the field of clinical application or pharmacy, the term “composition” usually has the same meaning as “pharmaceutical composition”.

  The actual dose level of odontogenic stem cells in the pharmaceutical composition or composition of the invention can be varied to be effective so that the particular recipient, i.e. the patient, receives good treatment or prevention. The dose level depends on the specific stem cell activity, route of administration, disease severity, therapeutic process of the disease or condition, treatment or procedure process of formation or repair (and manufacturing, regeneration, culture, etc.), disease state of the patient being treated Or it is selected according to medical history. However, in practice in the art, the stem cell dose and duration of application are gradually increased starting from below the level required to obtain the required therapeutic effect until the desired effect is obtained. Thus, in the case of the present invention, those skilled in the art can determine specific doses that apply to specific situations without the need for specific qualifications, based on the information provided by the present invention. There is no limitation. In particular, it may refer to a specific amount used in embodiments of the present invention to determine the amount used in any case.

  The odontogenic stem cells of the present invention can be administered alone (ie, in their original form) or in a pharmaceutical composition. The pharmaceutical compositions of the present invention can be formulated in a variety of suitable dosage forms depending on the route of administration. One or more physiologically acceptable carriers, excipients and auxiliaries can be used for odontogenic stem cells to be processed into a pharmaceutical formulation. The appropriate formulation form depends on the choice of route of administration that can be produced according to general knowledge in the field. In a plan of an embodiment of the present invention, odontogenic stem cells are present in a compatible medium (eg, saline, such as 0.9% normal saline). In one embodiment of the invention, odontogenic stem cells are present in a compatible medium and stored at low temperatures, for example, stored in a refrigerator, freezer, or other situation, optionally before use. Redissolved in a form applicable to the intended use.

  The present inventor has succeeded in confirming the therapeutic effect of odontogenic stem cells derived from different sources in the repair of periodontal bone tissue and soft tissue defects and other bone tissue damage, and has developed a powerful source of seed cells. Provided evidence. At the same time, it proves that the therapeutic effect of HGF genetically modified odontogenic stem cells in the repair of periodontal and soft tissue defects and other bone tissue damage is superior to odontogenic stem cells themselves, This showed that HGF and odontogenic stem cells play a synergistic role in the repair effect.

FIG. 1 shows the establishment of an experimental periodontal disease minipig model. A: Teeth before model establishment and their CT images. B: Teeth and their CT images 28 days after the periodontal disease model was established by establishing a periodontal disease model. This demonstrates the successful establishment of an experimental periodontal animal model.

FIG. 2 shows the effect of odontogenic stem cells on the treatment of experimental periodontal disease in minipigs. A: CT image of control group teeth before treatment. B: CT image of control group teeth 3 months later. C: CT image of anterior teeth before treatment with Ad-HGF modified DPSC suspension. D: CT image of anterior teeth 3 months after treatment with Ad-HGF modified DPSC suspension. E: CT image of anterior teeth before treatment with DPSC suspension. F: CT image of anterior teeth 3 months after treatment with DPSC suspension. G: CT image of anterior teeth before treatment with Ad-HGF modified DPSC + PDLSC suspension. H: CT image of anterior teeth 3 months after treatment with Ad-HGF modified DPSC + PDLSC suspension. I: CT image of anterior teeth before treatment with DPSC + PDLSC suspension. J: CT image of anterior teeth 3 months after treatment with DPSC + PDLSC suspension. K: General dental image of the control group after 3 months. L: General dental image 3 months after treatment with Ad-HGF modified DPSC suspension. M: General dental image 3 months after treatment with DPSC suspension. N: General dental image 3 months after treatment with Ad-HGF modified DPSC + PDLSC suspension. O: General dental image 3 months after treatment with DPSC + PDLSC suspension.

FIG. 3 shows periodontal clinical loss of periodontal attachment (AL), gingival bleeding index (SBI), and periodontal pocket depth (PD) in minipig odontogenic stem cell treated and control groups. The HGF-DPSC injection group represents a group injected with HGF-modified dental pulp stem cells. The DPSC injection group represents a group injected with unmodified dental pulp stem cells. The HGF-DPSC + PDLSC injection group represents a group injected with a mixture of HGF-modified dental pulp stem cells and HGF-modified periodontal ligament stem cells. The DPSC + PDLSC injection group represents a group injected with a mixture of dental pulp stem cells and periodontal ligament stem cells. The control group represents a group treated with physiological saline.

FIG. 4 shows changes in hematological indices of hemoglobin (HB), leukocytes (WBC), erythrocytes (RBC) and platelets (PLT) in the minipig odontogenic stem cell treatment group and the control group. The HGF-DPSC injection group represents a group injected with HGF-modified dental pulp stem cells. The DPSC injection group represents a group injected with unmodified dental pulp stem cells. The HGF-DPSC + PDLSC injection group represents a group injected with a mixture of HGF-modified dental pulp stem cells and HGF-modified periodontal ligament stem cells. The DPSC + PDLSC injection group represents a group injected with a mixture of dental pulp stem cells and periodontal ligament stem cells. The control group represents a group treated with physiological saline.

FIG. 5 shows changes in hematological indices IgA, IgG, IgE, IgM in minipig odontogenic stem cell treated and control groups. The HGF-DPSC injection group represents a group injected with HGF-modified dental pulp stem cells. The DPSC injection group represents a group injected with unmodified dental pulp stem cells. The HGF-DPSC + PDLSC injection group represents a group injected with a mixture of HGF-modified dental pulp stem cells and HGF-modified periodontal ligament stem cells. The DPSC + PDLSC injection group represents a group injected with a mixture of dental pulp stem cells and periodontal ligament stem cells. The control group represents a group treated with physiological saline.

FIG. 6 shows a CT image 2 weeks after the acute injury of the mouse femur. CON represents the blank control group. MSC represents a human dental pulp stem cell group. E-MSC represents an HGF genetically modified human dental pulp stem cell population.

Embodiment Implementation of the plan of the present invention will be described in detail in the following example. However, one of ordinary skill in the art will appreciate that the following examples are merely used to illustrate the present invention and should not be considered as limiting the scope of the invention. Let's go. Depending on conventional conditions or conditions recommended by the manufacturer, it may be performed under the conditions of more detailed embodiments. Even reagents or equipment not presented can be used by purchasing conventional products.

  αMEM culture medium was purchased from GIBCO USA. Its main components are sodium pyruvate, L-valine, alanine, linoleic acid, L-arginine hydrochloride ammonia acid, ascorbic acid, L-asparagine, biotin, L-aspartic acid, calcium D-pantothenate, L-cysteine Hydrochloride, folic acid, L-cysteine hydrochloride, inositol, L-glutamine, nicotinamide, L-glutamic acid, choline chloride, glycine, pyridoxine hydrochloride, L-histidine hydrochloride, riboflavin, L-isoleucine, thiamine hydrochloride, L- Examples include leucine, vitamins, and L-lysine hydrochloride.

Example 1: Isolation, culture, proliferation, and genetic modification of odontogenic stem cells (1) Isolation and culture of stem cells Isolation of gingiva, periodontal ligament, apex by tissue culture method or enzyme digestion method Cultured until the third generation. Odontogenic stem cells were identified as mesenchymal stem cells by detection of cell surface markers and osteogenesis and adipogenic differentiation. The actual isolation and culture method is as follows.

Periodontal ligament stem cell isolation and culture method:
Under anesthesia, the impact is applied to remove the human third molar, or the teeth are removed for orthodontics, then the removed teeth are placed in a centrifuge tube with sterile PBS, and antibiotics are added immediately, Periodontal ligament stem cells were separated over 12 hours. Gently exfoliate periodontal from central 1/3 of root, wash repeatedly with PBS, minced as finely as possible, put into 3 mg / ml type I collagenase and 4 mg / ml dispase solution, 0.5 to 1 hour at 37 ° C Digested by water bath, sieved through 70 μm cell sieves, collected cells, then centrifuged at 1000 rpm for 10 minutes and suspended in a single cell suspension with the appropriate amount of medium. Cells are cultured in 10-cm culture dishes in α-MEM medium (containing 10% fetal calf serum, 2 mmol / L glutamine), cultured at 37 ° C., 5% CO ₂ , and then cultured every 3-5 days Changed. The state of cell proliferation was observed every day under an inverted microscope. After 1-2 weeks, the cloned cells were digested with 0.25% trypsin and passaged to the next generation.

Methods for isolation and culture of dental pulp stem cells:
Under anesthesia, the impact is applied to remove the third molar, or the tooth is removed for orthodontic treatment, and then the removed tooth is placed in a centrifuge tube with sterile PBS, antibiotics added immediately, and the tooth Circumferential ligament stem cells were separated over 12 hours. Gingival tissue obtained by breaking the crown is repeatedly washed with PBS, minced as finely as possible, placed in 3 mg / ml type I collagenase and 4 mg / ml dispase solution, and bathed at 37 ° C. for 0.5-1 hour. Digested. Cells were harvested by sieving at 70 μm, then centrifuged at 1000 rpm for 10 minutes and suspended in a single cell suspension with the appropriate amount of media. Cells are cultured in 10-cm culture dishes in α-MEM medium (containing 10% fetal calf serum, 2 mmol / L glutamine), cultured at 37 ° C., 5% CO ₂ , and then cultured every 3-5 days Changed. The state of cell proliferation was observed every day under an inverted microscope. After 1-2 weeks, the cloned cells were digested with 0.25% trypsin and passaged to the next generation.

Methods for isolating and culturing root apical stem cells:
Under anesthesia, the hypoplastic human third molar root was removed, cut from the tip of the apex, washed repeatedly with PBS, minced, 3 mg / ml type I collagenase and 4 mg / ml dispase solution Digested by water bathing at 37 ° C for 0.5-1 hour, collect cells through 70 μm cell sieve, then centrifuge at 1000 rpm for 10 minutes, single cell suspension with appropriate amount of medium Floated on. Cells are cultured at 37 ° C., 5% CO ₂ in a 25 cm ² culture bottle of α-MEM medium (containing 15% fetal calf serum, 2 mmol / L glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin). After culturing, the ground culture was changed every 2-3 days. The state of cell proliferation was observed every day under an inverted microscope. When cells grew to 80% confluence, the cloned cells were digested with 0.25% trypsin and passaged to the next generation (1: 2). Flow cytometry was used to detect STRO-1, CD90, and CD146 surface markers for stem cell identification, and stem cells were obtained from the apex (SCAP).

  Odontogenic stem cells obtained from the above isolation and culture methods were examined for cell surface markers, osteogenesis and adipogenic differentiation (Perry BC, Zhou D, Wu X, Yang FC, Byers MA, Chu TM, Hockema JJ , Woods EJ, Goebel WS.Collection, cryopreservation, and characterization of human dental pulp-derived mesenchymal stem cells for banking and clinical use.Tissue Eng Part C Methods. 2008; 14 (2): 149-156; Pittenger MF, Mackay AM Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284 (5411): 143-147), and confirmed to be mesenchymal stem cells.

(2) In vitro modification of odontogenic stem cells 10,000 seeded odontogenic stem cells are cultured per square centimeter of culture area for 24 hours, and 150 MOI AD-HGF is added to the odontogenic stem cells (patent "a" AD-HGF preparation method according to recombinant adenovirus and its application in the treatment of myocardial ischemia “patent no. ZL 1129209.1”), 48 hours later, HGF genetically modified odontogenic stem cells were obtained.

  Odontogenic stem cells prepared as described above and in vitro modified odontogenic stem cells were used in the following examples.

Example 2: Therapeutic effect of odontogenic stem cells on periodontal bone and soft tissue defects First, a five-finger minipig experimental periodontal disease model was established. Thereafter, it was confirmed by CT images and clinical examination that a periodontal disease model was established, and the cell suspension was injected into the periodontal bone defect site using cell membrane transplantation and cell suspension injection techniques. Treatment of periodontal bone and soft tissue defects, pathological changes in periodontal tissues, changes in hematological indicators (blood routine, blood biochemicals, and immunoglobulin proteins), changes in inflammatory factors, from treatment Observations were made after 3, 7, 14, 28 and March. As examples for demonstrating the therapeutic effect of odontogenic stem cells on periodontal bone and soft tissue defects, dental pulp stem cells and periodontal ligament stem cells are shown below.

(1) Establishment of a miniature pig experimental periodontal disease model An experimental periodontal disease model was established using silk ligation and bone damage methods. Six 14-month-old minipigs were selected for the control group, dental pulp stem cell and periodontal ligament stem cell suspension injection group, and dental pulp stem cell suspension injection group. Each 2.3 mm × 5 mm × 7 mm defect (FIG. 1B) was created on the mesial surface of the first molar mandible, and 4.0 silk was attached to the defect and removed 10 days later. Clinical indices (plaque index, gingival bleeding index, periodontal pocket depth, and clinical periodontal loss) and image observation were performed before surgery, 6 weeks and 3 months after surgery. FIG. 1 shows that the establishment of an experimental periodontal disease model for minipigs was successful.

(2) Treatment with odontogenic stem cells for porcine bone and soft tissue defects caused by experimental periodontal disease
1 × 10 ⁷ MSC cell suspension and saline as a control were injected into the bone defect site of minipig periodontitis 4 weeks after surgery using cell suspension injection technique. The MSC cell suspension was a 9: 1 mixture of dental pulp stem cells and periodontal ligament stem cells.

(3) Results of treatment with odontogenic stem cells Three months after treatment with odontogenic stem cells modified with HGF, recovery of periodontal bone and soft tissue defects was significantly better than in the control group . A CT thin-layer coronary scan of the first molar clearly showed a newly formed bone image, but not in the control group. All specimens showed good repair and mild gingival swelling in the periodontal tissue of the odontogenic stem cell treatment group, whereas the control group had obvious gingival swelling and depression (eg, 2A, B, E, F, I, J, K, M, O).

  Clinical indicators (gingival crevice bleeding index, periodontal pocket depth, and loss of clinical periodontal attachment) of periodontal tissues injected with odontogenic stem cell suspension were better than controls (Figure 3).

Example 3: Therapeutic effect of HGF-modified odontogenic stem cells on repair of periodontal bone and soft tissue defects First, a minipig experimental periodontal disease model was established. Thereafter, it was confirmed by CT images and clinical examination that a periodontal disease model was established. The cell suspension was injected into the periodontal bone defect site. Periodontal bone and soft tissue defect repair and periodontal pathological changes were observed 3 days, 7 days, 14 days, 28 days and 3 months after treatment, respectively. As examples for demonstrating the therapeutic effects of HGF-modified odontogenic stem cells on periodontal bone and soft tissue defects, dental pulp stem cells and periodontal ligament stem cells are shown below.

(1) Establishment of a miniature pig experimental periodontal disease model Same as Example 2

(2) Treatment with HGF-modified odontogenic stem cells in a porcine experimental periodontal disease model
1 × 10 ⁷ HGF-modified dental pulp stem cell suspension and saline as a control were injected into the minipig periodontal bone defect site using cell suspension injection technique. It was divided into a control group, an HGF-modified dental pulp stem cell and periodontal ligament stem cell mixture injection group (two types of cells were modified), an HGF-modified dental pulp stem cell suspension injection group, and an unmodified odontogenic stem cell treatment group. The ratio of the number of dental pulp stem cells to periodontal ligament stem cells in the HGF-modified dental pulp stem cell and periodontal ligament stem cell mixture injection group was 9: 1.

(3) Results of treatment with HGF-modified odontogenic stem cells Treatment of HGF-modified odontogenic stem cells significantly recovered periodontal bone and soft tissue defects compared to control and unmodified cells It was very good. A median CT laminar coronary artery scan of the first molar clearly shows the newly formed bone image in the odontogenic stem cell group, and the newly formed bone is moderately shown in the unmodified group, Not shown in the control group. From all specimens, periodontal tissues in the HGF-modified odontogenic stem cell treatment group showed good repair, no gingival swelling was observed, and periodontal tissues in the odontogenic stem cell treatment group showed good repair and mildness In the control group, clear gingival swelling and depression were observed (eg, FIGS. 2B, C, D, G, H, K, L, N).

  Clinical indicators (gingival crevice bleeding index, periodontal pocket depth, and clinical periodontal attachment loss) of periodontal tissue treated with HGF-modified odontogenic stem cell suspension compared to control group It was good (FIG. 3).

  Furthermore, local injection of HGF genetically modified human odontogenic stem cell suspensions by hematology index (FIG. 4) and immunological detection index (FIG. 5) And it was shown that no significant immunological changes occurred.

  Other sources of HGF-modified odontogenic stem cells had similar therapeutic effects.

  These results indicate that the periodontal soft tissue and bone defect repair in the HGF-modified odontogenic stem cell treatment group is superior to that in the unmodified odontogenic stem cell group. HGF-modified odontogenic stem cells are not HGF-modified, because the animal inflammatory response of the HGF-modified odontogenic stem cells is superior to that of the control group and the unmodified odontogenic stem cells It was shown to have a therapeutic effect superior to odontogenic stem cells.

  According to clinical indicators and imaging results, both unmodified human odontogenic stem cells and HGF-modified odontogenic stem cells successfully repaired periodontal bone defects in a minipig experimental periodontal disease model, and blank controls It was shown that it was much better than.

  Because hematological studies showed that blood routine, biological, immunoglobulins, and immunological indicators did not change significantly in all groups before and after treatment, human odontogenic stem cells It is suggested that no significant change occurs with the injection of. These indicated that there were no inflammatory lesions, liver and kidney damage, immediate or delayed humoral immune response, and cellular immune rejection. The results of human odontogenic stem cell injection studies in the treatment of periodontal disease provide a powerful experimental basis for expanding the range of seed cell sources.

  In summary, odontogenic stem cells have a powerful therapeutic effect on the repair of periodontal bone and soft tissue defects, so that odontogenic stem cells can be used in tissue engineering for periodontal tissue regeneration. It becomes possible and suggests that a promising effect can be produced. HGF-modified odontogenic stem cells can express HGF locally within the damaged site of periodontal tissue and secrete various cytokines that enhance the effects of odontogenic stem cells, thereby treating periodontal bone defects Can exert biological anti-inflammatory effects.

Example 4: Repair of acute femoral damage (fracture) in mice (1) Establishment of femoral shaft fracture model in C57 mice (5-6 weeks) Experimental mice were treated with 10% / 2.5% pentobarbital sodium. The rats were injected intraperitoneally at a dose of kg, deeply anesthetized, the right hind limb was shaved, the right knee was bent 90 degrees, fixed in the supine position, disinfected with iodophor and covered with a sterile towel.

  (2) Make a 1cm longitudinal incision on the outside of the right knee, expose the distal femur and quadriceps tendon, push the quadriceps tendon inward, and complete the internal and external intercondylar grooves It was exposed and a 0.45 mm diameter stainless steel needle was inserted into the bone marrow and the interior was fixed with an intramedullary needle. The needle handle was cut and the end of the needle was embedded in the skin, after which the wound was closed.

  (3) The mouse moved to the bump model set of the bump test table. The internal fixed limb was placed at the flitch location, and then a weight of 17cm to 500g (adjusted appropriately according to the mouse's body shape) was dropped on the leg, and the femur was broken.

  (4) X-ray examination of fracture model under anesthesia

  (5) The mouse was resuscitated and moved to the animal room. On day 2, a local injection of stem cells was made.

2. Local injection of stem cells (1) Mice were divided into 3 groups of 3 mice each: blank control group (CON), human odontogenic stem cell injection group (MSC group), and HGF genetically modified human Dental pulp cell injection group (E-MSC group).

(2) The cell concentration of the MSC group and the E-MSC group was 5 × 10 ⁵ /0.4 ml, and the blank control group was 0.9% saline.

  (3) Mouse fracture site was disinfected locally with iodophor

  (4) The assistant fixed the mouse and injected stem cells into the fracture site from the center of the femoral shaft using a 1 ml syringe with 0.4 ml of cell solution.

3. Observation of fracture repair index Fracture repair was observed by CT 2 weeks after the injection of stem cells.

4). Results See Figure 6 for CT results. It can be seen that the control group had a large number of bone callus after 2 weeks and no clear bone repair was performed. The MSC group had a certain amount of bone callus and partial bone repair after 2 weeks. In the E-MSC group, most of the bone callus was resorbed after 2 weeks and the bone was basically repaired.

  While specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that all details and modifications and substitutions are within the scope of protection of the present invention based on all statements disclosed to the public. You will understand that. The full scope of the invention is determined by the appended claims and their equivalents.

Claims (15)

  Use of odontogenic stem cells in the preparation of a product for treating periodontal disease, repairing defects in periodontal bone or soft tissue and / or promoting regeneration of periodontal tissue.   Use of odontogenic stem cells in the preparation of a product for treating acute and chronic bone tissue damage (eg fractures) or bone tissue defects or to promote bone tissue repair.   The said odontogenic stem cell is at least one selected from the group consisting of dental pulp stem cells, deciduous deciduous dental pulp stem cells, periodontal ligament stem cells, and apical stem cells, for example, two types. use.   A genetically modified odontogenic stem cell into which an exogenous hepatocyte growth factor gene is introduced and the exogenous hepatocyte growth factor is expressed.   The genetically modified odontogenic stem cells are at least one selected from the group consisting of dental pulp stem cells, deciduous deciduous dental pulp stem cells, periodontal ligament stem cells, and apical stem cells, for example, two types. 4. The genetically modified odontogenic stem cell according to 4.   The genetically modified odontogenic stem cell according to claim 4 or 5, wherein the exogenous hepatocyte growth factor gene is introduced into the odontogenic stem cell by an adenovirus or an adeno-associated virus vector.   7. Any one of claims 4-6 in the preparation of a product for treating periodontal disease, repairing a periodontal bone or soft tissue defect and / or promoting regeneration of periodontal tissue. Use of the genetically modified odontogenic stem cells described.   7. Genetics according to any one of claims 4 to 6 in the preparation of a product for treating acute and chronic bone tissue damage (e.g. fractures) or bone tissue defects or for promoting repair of bone tissue. Use of engineered odontogenic stem cells. Treat periodontal disease, repair periodontal bone or soft tissue defects, promote periodontal tissue regeneration, treat acute and chronic bone tissue damage (eg fractures) or bone tissue defects And / or a composition comprising an effective amount of odontogenic stem cells and optionally a pharmaceutically acceptable carrier or excipient used to promote bone tissue repair,
Here, preferably, the odontogenic stem cells are at least one selected from the group consisting of dental pulp stem cells, stem cells collected from human deciduous deciduous dental pulp, periodontal ligament stem cells or stem cells at the root of the root, for example, two types Said composition.   A composition comprising an effective amount of a genetically modified odontogenic stem cell according to claims 4-6 and optionally a pharmaceutically acceptable carrier or excipient.   To treat periodontal disease, to repair periodontal bone or soft tissue defects, to promote periodontal tissue regeneration, to treat acute and chronic bone tissue damage (eg fractures) or bone tissue defects The use of hepatocyte growth factor in the preparation of a product to promote and / or promote bone tissue repair.   A method for preventing and / or treating periodontal disease, repairing a periodontal bone or soft tissue defect and / or promoting periodontal tissue regeneration, comprising an effective amount of claims 4 to 4. A method comprising administering a genetically modified odontogenic stem cell according to any one of claims 6 or a composition according to claim 9 or 10 to a subject in need thereof.   7. A method for preventing and / or treating acute and chronic bone tissue damage or bone tissue defect or for promoting bone tissue repair, wherein the effective amount is any one of claims 4-6. A method comprising administering a genetically modified odontogenic stem cell or a composition according to claim 9 or 10 to a subject in need thereof.   To prevent and / or treat periodontal disease, repair periodontal bone or soft tissue defects, promote periodontal tissue regeneration, prevent and / or prevent acute and chronic bone tissue damage or bone tissue defects Or odontogenic stem cells used to treat and / or promote bone tissue repair.   To prevent and / or treat periodontal disease, repair periodontal bone or soft tissue defects, promote periodontal tissue regeneration, prevent and / or prevent acute and chronic bone tissue damage or bone tissue defects The genetically modified odontogenic stem cell according to any one of claims 4 to 6, used for treatment and / or to promote bone tissue repair.

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